GATA-1 and heme regulate the erythroid cell transcriptome.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE74371 | GEO | 2016/01/07
SECONDARY ACCESSION(S): PRJNA299837
REPOSITORIES: GEO
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Project description:Alas2 gene encodes the rate-limiting enzyme in heme biosynthesis. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements strongly reduced GATA-1-induced Alas2 transcription, heme biosynthesis, and GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing Alas2 function in Alas2 cis-element-mutant (double mutant) cells by providing its catalytic product 5-aminolevulinic acid (5-ALA) rescued heme biosynthesis and the GATA-1-dependent genetic network. We discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. G1E-ER-GATA-1 WT and double mutant cells were examined. Untreated WT, beta-estradiol-treated WT, beta-estradiol-treated double-mutant, and beta-estradiol/5-ALA-treated double-mutant cells were subjected to RNA-seq.
2016-01-07 | E-GEOD-74371 | biostudies-arrayexpress
Project description:CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements in G1E-ER-GATA1 cells strongly reduced GATA-1-induced Alas2 transcription, heme biosynthesis, and GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing Alas2 function in Alas2 cis-element-mutant (double mutant) cells by providing its catalytic product 5-aminolevulinic acid (5-ALA) rescued heme biosynthesis and a subset of GATA-1-dependent genetic network. Using the same system, we discovered a GATA-1- and heme-dependent circuit that regulates chromatin accessibility during erythroid maturation.
2018-05-30 | GSE114996 | GEO
Project description:X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Apart from pyridoxine-responsive patients, treatment for XLSA is mainly supportive. Female XLSA often represents a later onset of severe anemia, mostly due to the acquired skewing of X-chromosome inactivation. We successfully generated active wild-type and active mutant ALAS2 induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and two daughters in a family with pyridoxine-resistant XLSA due to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with active wild-type iPSC lines. Most of the active mutant erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. Additionally, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed a significantly lower expression in active mutant erythroblasts, recapitulating the maturation defect. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid (ALA), confirming the suitability of the cells for drug testing. The administration of a DNA demethylating agent, azacitidine, reactivated the silent wild-type ALAS2 allele in active mutant iPSC-derived HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.
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Project description:5-aminolevulinic acid (ALA) is the common precursor of all biological synthezised tetrapyrroles. Inhibition of ALA synthesis results in decreased amounts of chlorophylls, heme, siroheme and phytochrome. It was previously shown that 4 out of 5 Arabidopsis mutants uncoupling nuclear gene expression from the physiological state of the chloroplast are affected in plant tetrapyrrole biosynthesis. It is common to all four mutants to show a reduced ALA formation. We investigate the impact of reduced plastidic ALA synthesis on nuclear gene expression by inhibition of ALA formation using gabaculin and compare these effects with the gun4-1 mutant. Formation of 5-aminolevulinic acid was inhibited in Arabidopsis thaliana Col-0 seedlings by application of 10 µM gabaculin. Additionally the Arabidopsis gun4-1 mutant was investigated. Seedlings were etiolated for 3 days on MS medium in absence or presence of gabaculin and subsequently deetiolated for 6 hours. Total RNA was extracted and used for microarray hybridisation.
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Project description:5-aminolevulinic acid (ALA) is the common precursor of all biological synthezised tetrapyrroles. Inhibition of ALA synthesis results in decreased amounts of chlorophylls, heme, siroheme and phytochrome. It was previously shown that 4 out of 5 Arabidopsis mutants uncoupling nuclear gene expression from the physiological state of the chloroplast are affected in plant tetrapyrrole biosynthesis. It is common to all four mutants to show a reduced ALA formation. We investigate the impact of reduced plastidic ALA synthesis on nuclear gene expression by inhibition of ALA formation using gabaculin and compare these effects with the gun4-1 mutant.
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Project description:In order to investigate the function of heme in the regulation of gene expression, we herein examined variations in mRNA levels in ALA-treated cells from control conditions. A comprehensive anal- ysis by RNA sequencing showed marked changes in the expression of various genes. Among the different amounts of mRNA, we identified the novel heme-inducible protein, SRRD. The plant ho- mologue Sensitivity to Red Light Reduced (SRR1) was previously reported to be involved in the regulation of the circadian clock and phytochrome B signaling in Arabidopsis thaliana. We found that SRRD regulated not only heme biosynthesis, but also the expression of clock genes. The involvement of SRRD in the prolif- eration of cells was also demonstrated.
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Project description:We addressed changes in gene expression profile in response to ; reduced oxygen tension in wild type or heme depletion caused by ; deletion of heme biosynthesis gene hem1 Experiment Overall Design: I.Heme depletion.The hem1 delta strain was grown for 6 hrs in YPD media supplemented with 250 uM ALA. Cells were washed three times with water to remove excess ALA. Cells were then inoculated in duplicates into 15 ml cultures of Experiment Overall Design: YPD media that was supplemented either with or without ALA. Cultures were induced for 16 h before 5 ml of cells were harvested and washed Experiment Overall Design: three times in DEPC water. The culture density at the time of harvest Experiment Overall Design: was approximately OD600=0.5. Experiment Overall Design: II.Hypoxia. BY45742 strain was grown for 6 hrs in Experiment Overall Design: YPD medium and then inoculated in 15 ml the same medium and grown Experiment Overall Design: under normoxic or hypoxic conditions for 12 hrs. To create hypoxic Experiment Overall Design: conditions, cultures were incubated in an anaerobic chamber using a Experiment Overall Design: BBL-GasPak (BD Biosciences).
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Project description:5-aminolevulinic acid (ALA) is one of the natural amino acids and a product of the first heme synthesis pathway in mitochondria. Recently, a supplement containing ALA is available in Japan and used for the purpose of the enhancement of ATP synthesis via mitochondrial activity. In humans, the immunomodulatory effect of ALA has attracted attention as a new function of ALA, and its application to cancer, inflammatory disease, and autoimmune diseases are being investigated. In the present study, to evaluate the effects of ALA on canine immunity, we performed in vitro studies using peripheral blood mononuclear cells (PBMC) from healthy dogs. Heme oxygenase-1 protein was expressed in Madin-Darby Canine Kidney cells and PBMCs treated with 5-ALA and ferrous sodium citrate (SFC), which showed that ALA works in dogs as well as humans. When PBMCs were stimulated with concanavalin A (ConA), the addition of ALA resulted in a significant increase in interferon-gamma (IFN-γ) produced by ConA-stimulated PBMCs. A comprehensive genetic alteration using next-generation RNA sequences (RNA-seq) was performed. From the result of RNA-seq, ALA enhanced the gene expression of T cell immunity including Th1, Th2, and Th17 subsets. In particular, the IL-17 signaling pathway was significantly upregulated. Then, we confirmed that ALA promoted the production of interleukin (IL)-17A in ConA-stimulated PBMCs. Together, these findings reveal that ALA promotes heme synthesis in mitochondria and enhances ConA-induced T cell immune responses in canine PBMCs.
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Project description:In mice lacking the heme exporter, FLVCR, differentiation fails at the CFU-E/proerythroblast stage from excessive heme and reactive oxygen species. We show that Flvcr1-deleted CFU-E/proerythroblasts have low GATA1 mRNA and GATA1-target gene mRNAs along with increased ribosomal protein mRNAs as a direct result of increased heme. Thus heme increases ribosomal protein transcription when globin production needs to be increased, and when heme is excessive, GATA1 is reduced allowing for normal termination of erythroid differentiation. This demonstrates that heme and GATA1 are co-master regulators of erythroid differentiation.
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Project description:Deciphering cellular proteomes is critical for our understanding of erythropoiesis. To better comprehend commonly used models of erythropoiesis, we obtained absolute quantification of proteins expressed in cultured primary murine bone-marrow derived erythroblasts throughout erythroid differentiation. These data were compared to proteomes from Friend murine erythroleukemia (MEL), G1ER, and MEDEP cells. Overall, more than 7200 proteins were quantified in at least one of these models of murine erythropoiesis. Protein expression during differentiation of MEDEP cells was most similar to that of differentiating primary murine erythroid cells, while proteomes of Friend MEL and G1ER cells were more distantly related. Comparison of the proteomes of murine and human erythroblasts revealed species-linked variability, but these differences were not as dramatic as those observed comparing human and murine transcriptomes during erythroid differentiation. To functionally validate the differences between transcriptional and translational control, heme synthesis was inhibited in MEDEP cells and the effects on the transcriptome and the proteome examined. While heme deficiency had minimal global effect on the cellular proteome, globin proteins were significantly down regulated, supporting observations heme regulation of globin synthesis is tightly regulated at the protein level to avoid deleterious over production of globin chains. As predicted, heme deficiency led to up regulation of -aminolevulinic acid synthase 2 (Alas2) protein. Interestingly, Alas1 was upregulated at both the transcriptional and protein levels. Characterization of cellular proteomes during erythropoiesis has potential to yield insight into mechanistic principles of human disease, as well as reveal novel therapeutic targets.
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